A wide variety of chemical moieties (‘payloads’) have been covalently attached to enzymes, antibodies, and other large, polypeptides or proteins, to form conjugates. The payloads may be used to locate the protein to which they are attached (e.g., labels), to modify the physicochemical properties or stability of the protein (e.g., PEGylation), to enable the protein to be attached to another molecule or protein (coupling groups for connecting the conjugate to another compound or another conjugate), or to modify the function or activity of the payload or the protein (e.g., vaccine conjugates). The protein may also act as a carrier to deliver the attached payload to a particular tissue or cell type, such as in antibody-drug conjugates (ADCs). Classes of payloads that can be usefully linked to proteins include detectable moieties (labels), anchoring moieties to attach the protein to a surface or another compound, antigens that elicit an immune response when conjugated to a protein, coupling groups that react readily with a chemically complementary coupling partner (thus connecting the protein to another entity), and therapeutic moieties such as cytotoxins and other bioactive agents. Attaching these diverse structures to proteins in a controlled and reproducible fashion is often critical for the conjugates to function correctly. Thus there is a need for a variety of methods to attach many types of payloads to many different proteins or polypeptides.
A number of methods have been developed for attaching payloads to proteins to form protein conjugates. See, e.g., Sletten, E. M. and Bertozzi, C. R. Angew. Chem. Int. Ed. 2009, 48, 6974-6998; Basle', E.; Joubert, N.; and Pucheault, M. Chemistry & Biology 2010, 17, 213-227. In some protein conjugates, the method by which the protein and payload are connected may have undetectable impact on the activity or relevant properties of the conjugate; in other instances, the nature of the linkage between protein and payload can significantly affect the activity or properties of the conjugate. Sometimes it is critical to control the number of payload moieties per protein, for example, or to control the precise location where payloads are attached so they do not interfere with functions of the protein. ADCs, for example, require the protein to recognize and bind to surface structures on targeted cells to impart selectivity, so a payload must not be positioned to interfere with binding of the antibody to the surface structures (antigen) that the antibody must recognize. See, e.g., Expert Opin. Biol. Ther. 2012, 12, 1191-1206; Toxins 2011, 3, 848-883; and Laurent Ducry Drug Delivery in Oncology: From Basic Research to Cancer Therapy, 1st Edition. Wiley-VCH Verlag GmbH & Co. KGaA. 2012, 12, 355-374.
Most methods for attaching payloads to proteins involve adding a linking chemical structure (linker) between the protein and the particular payload of interest. The linker provides a way to connect the payload of interest to the protein using available functional groups on each moiety. The linker often allows the distance between payload and protein to be modulated, and may also include a cleavable portion that can be lysed or degraded in vivo to release the payload from the protein where release is important for the payload to achieve its objectives. For example, in ADCs, it may be critical for the conjugate to break down and release the payload at a location where it can have a desired effect. Because of the diverse types of protein-payload conjugates that place different demands on the manner in which the payload and protein are connected, there is a continuing need for novel methods to link payloads to proteins consistently and efficiently.
The most common methods for forming protein conjugates rely upon the chemical reactivity of certain amino acids that occur naturally in many natural proteins: lysine and cysteine are often used because they provide a reactive site for connecting the payload to the protein. Lysine has a free amine group that can react with a suitable electrophilic functionality on a linking group or payload, and cysteine can react through its free sulfhydryl group. However, relying on these naturally occurring reactive sites can be complicated: when there are too many or too few of the particular type of amino acid in a protein of interest, for example, it becomes difficult to get just the right ‘loading’ of payload on the protein. The high abundance of lysine on protein surfaces makes site- and regio-selective conjugation difficult, and leads to heterogeneous products. In contrast, cysteines are comparatively rare, and exist mainly in disulfide-linked pairs in proteins. Conjugation at cysteine often requires reduction of a disulfide, followed by reaction with a conjugation reagent (e.g. maleimide) to label individual cysteines separately. Because this removes a disulfide linkage, the protein structure and stability might be undermined by this process.
Proteins also often have more lysines than the optimum number of payloads to be attached: adding enough payload moieties to occupy all of the availably lysines in order to ensure a consistent, homogenous product may add too many payload molecules for optimum efficacy. This can be avoided by using only some of the lysines for conjugation, but such partial or incomplete loading will generally provide a heterogeneous product, which can be problematic for a variety of reasons—in the case of Mylotarg™, the first commercialized ADC, for example, the heterogeneity of the ADC product seems likely to have contributed to the issues that led to a decision to withdraw the product from registration. Fuenmayor, et al., Cancers, vol. 3, 3370-93 (2011). Also, even when enough amino acid groups of a particular type (e.g., lysines) are present for optimal loading, some or all of them may be ‘buried’ inside the protein when the protein is in its solution conformation, rendering them effectively unavailable for conjugation, or making them ‘partially’ accessible which can also result in heterogeneity of the conjugate. Thus, while lysine can be a useful site for conjugation, in many situations it is not ideal.
The frequency of occurrence of cysteine in natural proteins is lower than that of lysine, and cysteine may be suitable for use as a site for conjugation where it is available in adequate numbers; where too few cysteines are present, one or more may be inserted by standard protein modification methods. However, it is often preferable to avoid modifying the sequence of the natural protein by inserting a cysteine; besides, surface-accessible cysteines in natural proteins are often positioned near other cysteines to form disulfides, which may be important for maintaining the protein's active conformation. While it is not difficult to convert a disulfide into two free cysteines by reducing the disulfide, doing so may disrupt the secondary or tertiary structure of the protein.
Some methods for attempting to insert a tether between cysteine residues formed by reducing a disulfide on a protein have been reported. One such method involves a sulfone-substituted methacrylate derivative. US2006/0210526. This method forms a reactive intermediate that requires an elimination step before cyclization, and the conditions for that multi-step process can result in incomplete formation of a linker (tether) between cysteines, and the reaction conditions can even cause protein denaturation. Another approach uses a maleimide derivative. WO2011/018613. However, the conjugate formed in this process suffers from stability problems because the Michael addition of the thiols on the maleimide is reversible. There is thus a need for improved methods to conjugate chemical moieties to proteins containing disulfide linkages to form protein conjugates. In particular, methods are needed that use the disulfide components (sulfhydryls) without giving up the conformation controlling effect of the disulfide, while also providing efficient conjugation, stability, and consistent payload/protein ratios. In addition, there is a need for stapling methods to hold proteins in a particular conformation (see, e.g., Expert Opin. Drug Discov. (2011) 6(9):937-963; Tetrahedron 55 (1999) 11711-11743) that also provide a means to conjugate the stapled protein with a payload. The present invention provides such methods.